1. Field of the Invention
This invention relates generally to systems for culturing cells. More particularly, the invention relates to a tri-methylamine (TMA) microcarrier bead system which serves as a microcarrier support for the culturing of anchorage-dependent cells.
2. Description of Related Art
The use of microcarrier supports to facilitate growth of biological cells has a long and varied history. Early systems for effecting such cell growth in useable quantities have included the well-known petri dish and flasks. Efforts intended to increase the quantity of cell production have resulted in the use of large trays. The overall effect achieved being the increase in the surface area on which the biological cells are grown, specifically anchorage-dependent cells. Some of these early cell-growth systems are still in use for applications where small-scale, high labor content cell culture systems will suffice, such as in hospital and university research units.
The state of the cell culturing art has continued to develop to the present day where there is an acute need for large scale, low labor content, commercial cell culturing systems which can achieve high rates of production. Ideally, the production rate should be flexible to accommodate batch and single order production.
At present, the cell culturing system which enjoys utilization in some 60% to 80% of the commercial market is the roller bottle system. Essentially, a roller bottle is a cylindrical container which is arranged to contain a small amount of nutrient media. In operation, the roller bottle is rotated slowly about its longitudinal axis, at about 1 to 3 rpm, whereby the nutrient media is continually caused to wet the entire interior surface of the bottle, on which cell growth is achieved. A plurality of such roller bottles are operated on a roller rack, the specific number thus rotated being responsive to the desired rate of overall cell production.
The remaining 20% to 40% of the commercial large scale market utilizes microcarrier systems. Although other techniques have been introduced in recent years, including hollow fibers, fiber bundles, cell growth cubes, and channeled ceramic cores, only microcarrier systems have the potential to achieve anchorage dependent cell growth at commercially advantageous production rates with low labor content. Achieving large scale in this manner is superior to achieving it by replication, as is the case with roller bottles and other known systems. In addition, microcarrier bioreactor systems are well-suited for automated large scale cultivation of anchorage dependent cells.
As is evident from Table 1 below, several microcarrier systems are presently commercially available. This table summarizes some of the characteristics of the commercially available microcarriers, including polystyrene, glass-coated polystyrene, and the novel TMA microcarriers of the present invention.
The British-based company, Amersham-Pharmacia-Biotech, was the first to introduce the microcarrier bead in the early 1970's. Subsequently, this company has acquired about a 90% share of the microcarrier bead market, world-wide.
The use of microcarrier beads as the microcarrier elements in anchorage dependent cell production systems requires the availability of bioreactors, support equipment, and a stirring system. The system elements interact with one another to maintain the cell-laden microcarrier beads in suspension in the nutrient media. Much of this type of equipment is commercially available, and the effort to develop and improve bioreactor systems for use with microcarrier beads has intensified.
Current microcarrier use in the large-scale cultivation of anchorage dependent cells. Large scale cultivation of anchorage dependent cells is done primarily for the production of vaccine strain viruses used in human and animal medicine. Genetically engineered biological production has recently begun use of large scale, cell culture technologies. Two additional uses, production of virus vectors for gene therapy and production of specific cell types for cellular therapies, will utilize the same technologies in the future. Nothing in the foreseeable future is likely to reduce the need for large quantities of anchorage dependent cells. In the past, roller bottles have been the most extensively used technology in large scale cell culture operations. More recently, other technologies including hollow fiber culture systems and microcarrier/bioreactor systems have replaced roller bottles in some applications. Both technologies have certain advantages over roller bottles which will likely make this trend continue. Hollow fiber reactors are useful for growing anchorage independent cells (e.g., mainly hybridomas for antibody production), but are not optimal for large-scale cell culture operations. Microcarrier/bioreactor systems offer the best alternative to roller bottles for the large-scale cultivation of anchorage-dependent cells with a low labor content.
Microcarrier development started in 1967 when van Wesel demonstrated that DEAE--dextran beads could be used as a substrate for the growth of anchorage-dependent cells in a suspension culture mode (Van Wezel (1967). Since that time, a number of different materials including glass, polystyrene plastic, acrylamide, solid collagen, porous collagen, cellulose and liquid fluorocarbons have been successfully used as microcarriers (Varani, et al. (1983); Nielson, et al. (1980); Obrenovitch, et al. (1982); Giard, et al., (1977); Gebb, et al., (1982)). In addition, microcarriers with one or more adhesive peptides attached to the surface through covalent or noncovalent linkages have been used (Keese, et al. (1983); Varani, et al. (1988); Varani, et al., (1986)). To be useful as a microcarrier, a material must have a surface chemistry which supports cell attachment and growth, and must not be toxic to the cells or to the elaborated products. The ideal microcarrier should have a diameter of approximately 75-225 .mu.m, although larger or smaller sizes (U.S. Pat. No. 5,114,855 (May 1992); J. Varani, S. Josephs and W. Hillegas, "Human Diploid Fibroblast growth in polystyrene microcarriers in aggregates", Cytotechnology, 22: 111-117 (1996)) have been used. The ideal density appears to be in the range of 1.02-1.05 g/cc, although lighter or heavier material may be better suited for certain applications. In addition to differences in surface chemistry, microcarriers made from one substance or another differ in such characteristics as rigidity, porosity and adsorptive capacity. Differences in handling characteristics, durability, shelf-life and ease of sterilization all distinguish one substance from another as does overall manufacturing costs. From the standpoint of commercial potential, all of these variables must be considered.
Although a large number of different types of material have been developed for use as microcarriers, only two types of microcarrier products are widely-used in the industry today. These are the i) dextran-based microcarriers (Cytodex I, DEAE-dextran; and Cytodex III, porcine collagen-coated dextran) made in Sweden and sold by Amersham-Pharmacia Biotech of the United Kingdom and ii) the coated polystyrene based microcarriers made in the United States by SoloHill. Microcarriers made by SoloHill have been successfully integrated into manufacturing processes in the United States, Europe and Japan. SoloHill makes a porcine collagen-coated polystyrene microcarrier bead, which is heavily used in the animal health industry to produce viral vaccines. Smaller amounts of Solohill's glass-coated polystyrene microcarriers have also found a use in industry, and an intense interest has developed in the recently-released ProNectin F*--coated polystyrene beads, largely because they are free of animal proteins. (*ProNectin F is a genetically engineered protein incorporating multiple copies of the cell attachment ligand (RGD) from fibronectin. It is available from Protein Polymer Technologies, Inc..)
However, the entire viral vaccine industry appears to be on the cusp of a revolution, in which virtually every existing protocol will come into question. This is due largely to concerns about bovine spongiform encephalopathy (BSE), the "mad cow" disease, in the food chain and the attendant effort to eliminate animal-derived proteins from manufacturing processes. One focus is on replacement of serum-containing culture media with fully synthetic media. However, as serum containing media are eliminated, there will be no tolerance for substrates containing bovine or porcine collagen or any other animal protein. As indicated above, most manufacturing procedures that use microcarriers use either the collagen-coated dextran beads (Amersham-Pharmacia Biotech) or the collagen-coated polystyrene beads produced by SoloHill. The push to eliminate animal proteins from vaccine manufacturing thus provides an opportunity for wide evaluation of a novel, animal protein-free substrate such as the TMA microcarriers. The European regulatory agency (EMEA), it should be noted, has already begun a strong effort to remove animal-derived proteins from biological product manufacturing. In the United States, the FDA issued an "advisory" on May 9, 1996 the inference of which is that manufacturers should move expeditiously to eliminate animal proteins from their processes stating "We strongly recommend you take whatever steps are necessary to assure yourselves and the public that, in the manufacture of FDA-regulated products intended for administration to humans, you are not using materials that have come from cattle born, raised, or slaughtered in countries where BSE is known to exist. FDA believes that immediate and concrete steps must be taken by manufacturers to reduce the potential risk of human exposure to, or transmission of, the infectious agent which causes BSE in cattle."
It would therefore be useful to develop a microcarrier system which does not contain animal proteins.